Dendritic Ca2+ Channels Characterized by Recordings from Isolated Hippocampal Dendritic Segments  Ege T Kavalali, Min Zhuo, Haruhiko Bito, Richard W Tsien  Neuron  Volume 18, Issue 4, Pages 651-663 (April 1997) DOI: 10.1016/S0896-6273(00)80305-0

Figure 1 MAP2, a Dendritic Marker Protein, Is Expressed in Isolated Dendritic Segments (A) A phase-contrast photomicrograph shows an isolated cell soma with a proximal dendrite (top) and soma-free dendrites (“dendrosomes”). Arrowheads indicate dendritic structures. (B) Phase-contrast view of a hippocampal neuronal culture at 14 days in vitro. (C) Immunofluorescent staining with anti-MAP2 (FITC) and anti-tau (Texas Red) antibodies of the same culture as in (B), visualized under an epifluorescence microscope using a triple-band filter set. Tau-positive (red) axonal segment is indicated with the arrow, whereas MAP2-positive (green) dendrites are indicated with arrowheads. (D) Phase-contrast view of an isolated cell soma with a few processes. (E) Immunofluorescent staining with anti-MAP2 (FITC)/anti-tau (Texas Red) of the same specimen. Most of the processes were MAP2-positive and were thus identified as dendrites (arrowhead). However, in a number of cases as shown here, one of the processes was strongly immunopositive for tau (arrow), suggesting that this one was of axonal origin. (F and G) Phase-contrast view (F) and immunostaining with MAP2 (FITC)–tau (Texas Red) (G) of a dendrosome. MAP2 positivity (in green) identified the dendritic origin of dendrosomes. In these experiments, the absence of staining with DAPI (in blue, used to visualize nuclei in [C] and [E]) verified the absence of nuclear contents in the dendrosomes. Scale bars, 20 μm. Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)

Figure 2 Postsynaptic Markers Are Expressed in Dendrosomes Expression of two postsynaptically enriched proteins, GluR1 ([A], [C], and [E]) and CaMKIIα ([B], [D], and [F]), were independently demonstrated with confocal microscopy. Specimens include soma-free dendrosomes ([A] and [B]), soma with an attached dendrite ([C] and [D]), and cultured hippocampal neurons ([E] and [F]). Immunoreactivities for GluR1 (green pseudocolor) and CaMKIIα (red pseudocolor) were both found in dendrosomes as well as in dendritic structures in a more native state, suggesting that the localization and expression of membrane channels and cytoplasmic proteins were retained during the preparation of the dendrosomes. Scale bars, 5 μm. Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)

Figure 3 Active Properties of Isolated Hippocampal Dendrites (A) A dendrosomal capacitative transient evoked by a depolarization from −80 to −70 mV. The transient could be fitted with a single exponential function (τ = 117 μs). (B) Inward Na+ and outward K+ currents evoked by a series of depolarizations (HP = −90 mV) to test potentials ranging from −30 mV to +10 mV in 10 mV increments. (C) Representative traces from an experiment in which the inward Na+ current was isolated by replacing the K+ in the pipette solution with Cs+. (D) Dendrosome APs elicited by current injection, compared with (E) spikes recorded from a cell soma without visible processes. In both cases, membrane potential was hyperpolarized to −90 mV with a steady conditioning current of ∼100 pA before application of a 50 pA depolarizing current pulse. (F) Block of dendrosome APs by application of 1 μM TTX. (G) Amplitudes of dendrosomal and somatic APs as measured from threshold (see text for details). Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)

Figure 4 Comparison of Ca2+ Currents in Dendrosomes versus Isolated Cell Somata (A and B) Ca2+ currents recorded from dendrosome (A) and cell body (B), evoked by 160 ms depolarizing pulses from HP = −90 mV to test potentials ranging from −50 through 0 mV as indicated. (C) Mean membrane capacitance values of dendrosomes and cell bodies without visible processes. (D) Current density versus voltage relations for dendrosomal (closed squares) and somatic (open circles) Ca2+ currents with 5 mM Ba2+ as the charge carrier (see text for details). Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)

Figure 7 Dendritic Currents in 2 mM Ca2+ (A and B) Representative Ca2+ currents from a dendrosome recording with 2 mM Ca2+ activated during a 80 ms depolarization from HP = −90 mV (A) or HP = −70 mV (B) to test levels as indicated. (C) Normalized peak current versus test potential relation for HP = −90 mV (closed circles) and HP = −70 mV (open circles) (n = 7). I–V curves were normalized with respect to the largest value of peak current amplitude detected from HP = −90 mV (typically at a test level of 0 or −10 mV). The reduction in peak current at test potentials between −50 and +20 mV was statistically significant (P < 0.05). (D) Deactivation kinetics of Ca2+ currents following 10 ms depolarizations to −40 and 0 mV (HP = −90 mV; sampling rate = 40 μs). Tail currents could be fitted with two exponentials with time constants of 0.16 and 1.44 ms. The smooth curve depicts slow exponential component. (E) Plot of peak tail current amplitude versus test potential from the same experiment as in (D). Boltzmann functions representing voltage dependence of slow component (solid curve) Aslow = 124.3/(1+exp[(−39.0−Vt)/10.7]) and for fast component (dashed) Afast = −85.7/(1+exp[(−18.8−Vt)/7.8]). Differences in Boltzmann slopes of plots in Figure 6E and Figure 7E are due to noise. Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)

Figure 7 Dendritic Currents in 2 mM Ca2+ (A and B) Representative Ca2+ currents from a dendrosome recording with 2 mM Ca2+ activated during a 80 ms depolarization from HP = −90 mV (A) or HP = −70 mV (B) to test levels as indicated. (C) Normalized peak current versus test potential relation for HP = −90 mV (closed circles) and HP = −70 mV (open circles) (n = 7). I–V curves were normalized with respect to the largest value of peak current amplitude detected from HP = −90 mV (typically at a test level of 0 or −10 mV). The reduction in peak current at test potentials between −50 and +20 mV was statistically significant (P < 0.05). (D) Deactivation kinetics of Ca2+ currents following 10 ms depolarizations to −40 and 0 mV (HP = −90 mV; sampling rate = 40 μs). Tail currents could be fitted with two exponentials with time constants of 0.16 and 1.44 ms. The smooth curve depicts slow exponential component. (E) Plot of peak tail current amplitude versus test potential from the same experiment as in (D). Boltzmann functions representing voltage dependence of slow component (solid curve) Aslow = 124.3/(1+exp[(−39.0−Vt)/10.7]) and for fast component (dashed) Afast = −85.7/(1+exp[(−18.8−Vt)/7.8]). Differences in Boltzmann slopes of plots in Figure 6E and Figure 7E are due to noise. Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)

Figure 5 L-, N-, and P/Q-Type Ca2+ Channels in Dendrosomes (A) Inhibition of dendritic Ca2+ currents by 1 μM nimodipine (n = 7). In this panel and others, the time course of block was obtained by pooling data from multiple dendrosomes; peak current amplitudes at 0 mV (HP = −80 mV) in individual experiments were normalized with respect to their control values before averaging. Traces on the right show current records from a representative experiment before and during application of nimodipine. (B) Application of 1 μM ω-CTx-GVIA (50 s) reveals a component of dendritic Ca2+ current carried by N-type Ca2+ channels (n = 5). (C) The time courses of inhibition of Ca2+ current by 20 nM (triangles) and 1 μM (circles; applied for 50 s) ω-Aga-IVA. Traces on the right show the dendritic Ca2+ currents before and during application of 20 nM and 1 μM ω-Aga-IVA, respectively, from two experiments. (D) Time course of inhibition of Ca2+ current by ω-CTx-MVIIC (5 μM). Note fast and slow components of block, presumably corresponding to inhibition of N- and P/Q-type Ca2+ channels, respectively. Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)

Fig. 6 Ca2+ Currents Spared by ω-CTx-MVIIC and Nimodipine (A) The time course of inhibition of Ca2+ current by 5 μM ω-CTx-MVIIC and 10 μM nimodipine combined (n = 5). These agents were applied while dendrosomes were depolarized to 0 mV (HP = −80 mV). At steady state, 20.3% ± 3.5% (n = 4) of the initial current remained. Inset, representative Ca2+ currents before and 3 min after exposure to MVIIC+nimodipine. (B) Current-voltage relationships of Ca2+ currents in hippocampal dendrosomes (n = 4) and cerebellar granule cells (n = 5) (HP = −90 mV). In pooling data, current amplitudes in individual experiments were normalized with respect to the largest peak current amplitude. (C and D) Representative traces from a hippocampal dendrosome (C) and a cerebellar granule cell (D), after application of ω-CTx-MVIIC+nimodipine as in (A). Ca2+ currents evoked by 80 ms depolarizations (HP = −90 mV). (E) Analysis of tail currents in dendrosomes reveals multiple components of Ca2+ current in the presence of ω-CTx-MVIIC+nimodipine. Inset, recordings of tail current (sampling rate = 40 μs), reflecting deactivation at HP = −90 mV following a 10 ms depolarization to indicated test levels. Smooth curves represent single exponential fits. The graph shows the voltage dependence of the amplitude of the slow and fast components of tail current with their respective Boltzmann fits, where Aslow = −49.0/(1+exp[(−42.7−Vt)/4.3]) (solid curve) and Afast = −93.1/(1+exp[(−5.7−Vt)/8.8]) (dashed curve). Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)

Figure 8 Neurotransmitter Modulation of Dendritic Ca2+ Currents (A) Inhibition of dendritic Ca2+ currents by activation of metabotropic glutamate receptors. Application of (1S, 3R)–ACPD (200 μM) caused a 43% decrease in the peak current in this particular experiment but only a 14% decrease when reapplied after blockade of N-type Ca2+ channels with ω-CTx-GVIA (1 μM). Superimposed current traces on the right show records taken before and during agonist application (a,b: before ω-CTx-GVIA; c,d: after ω-CTx-GVIA). (B) Inhibition of dendritic Ca2+ currents by activation of GABAB receptors. In this example, baclofen (50 μM) caused a 53% inhibition initially, but the inhibition was reduced to 26% after application of ω-CTx-GVIA. Traces marked as in (A). (C) Pooled data representing the inhibition by neurotransmitter receptor agonists, (1S,3R)–ACPD (ACPD), R(+)–baclofen (BAC), and somatostatin (SST), before and after the application of 1 μM ω-CTx-GVIA (see text for details). The asterisk indicates statistical significance (P < 0.05). Neuron 1997 18, 651-663DOI: (10.1016/S0896-6273(00)80305-0)